Process for preparing cephalosporanic acid derivatives using alpha-ketoacid derivatives

ABSTRACT

A process for preparing cephalosporanic acid derivatives comprises the steps of enzymatically converting a 3-thiolated cephalosporin C compound of formula III:— 
                 
 
     into a 3-thiolated-α-ketoadipyl-7-aminocephalosporanic acid derivative of formula IV:  
                 
 
     wherein R is a heterocyclic group comprising at least a nitrogen atom.  
     Compounds of formula IV are used in the preparation of cephalosporin C antibiotics and derivatives thereof.

[0001] The invention relates to a process for preparing 3-cephalosporinC derivatives which are used in the preparation of β-lactam antibiotics.In particular the invention relates to an enzymatic process for thepreparation of 3-thiolated derivatives of3-acetoxy-methyl-7-amino-ceph-3-em-carboxylic acid (3-thiolated-7-ACA)using α-ketoacid intermediates. The α-ketoacids or α-oxoacids areimportant biopharmaceutical compounds.

[0002] Oxoacids of essential amino acids are gaining importance asnutraceuticals (Pszcola, D E, Food Technol. 52, 30, 1998) as well astherapeutic agents for treating nitrogen accumulation disorders(Schaefer et al., Kidney Int. Suppl. 27, S136, 1989; Buto et al,Biotechnol. Bioeng. 44, 1288, 1994). Another important application isthe production of 7-amino cephalosporanic acid (Savidge, TA; InBiotechnology of Industrial Antibiotics, p 171, Marcel Dekker, New York,1984) from cephalosporin C(3-acetoxymethyl-7β-(D-5-amino-5-carboxypentanamido)ceph-3-em-4-carboxylic acid). The transformation can be carried out by aD-amino acid transaminase from Bacillus licheniformis ATCC 9945, whichconverts cephalosporin C with α-ketoacids into α-ketoadipyl-7-ACA andthe corresponding D-α-amino acid, as described in DE 3447023 (Hoechst).This conversion is a transamination, the amino group of cephalosporin Cbeing converted non-oxidatively into the keto group, without the releaseof hydrogen peroxide. However there is a low level of activity of thisenzyme, as described in EP 0315786.

[0003] Chemical methods for the preparation of 3-thiolated-7-ACAcephalosporanic acid derivatives are known (U.S. Pat. No. 3,367,933; BE718,824), however they have disadvantages such as low temperaturereaction conditions, the use of costly and toxic solvents or reagentsand chemical instability of intermediates which makes the processesdifficult on an industrial scale.

[0004] To overcome the drawbacks of the chemical route to 7-ACA,alternative enzymatic cleavage of cephalosporin C has been described.Direct one-step removal of the lateral 7′-aminoadipic side-chain ofcephalosporin C is possible by using specific cephalosporin acylases (FR2,241,557; U.S. Pat. No. 4,774,179; EP 283,248; WO 9512680; WO 9616174).These processes, however, are often not reproducible and arecharacterised by low yields and lengthy reaction times as described inU.S. Pat. No. 5,296,358. No industrial application of this technology(single-step conversion of cephalosporin C to 7-ACA) has been reportedat this time (Parmar et al, Crit. Rev. Biotechnol. 18, 1, 1998).

[0005] On the other hand, processes that transform the cephalosporin Cinto 7-ACA by means of two enzymatic steps are important from anindustrial point of view. The first stage consists of using a D-aminoacid oxidase (E.C. 1.4.3.3, hereinbelow indicated as DAAO) fromdifferent sources (Trigonopsis variabilis, GB 1,272,769; Rhodotorulagracilis, EP 0,517,200; or Fusarium solari M-0718, EP 0,364,275). DAAOoxidises the lateral D-5-amido-carboxypentanoyl chain of cephalosporin Cin the presence of molecular oxygen, to produce.7β-(5-carboxy-5-oxopent-amido)-ceph-3-em-carboxylic acid (orα-ketoadipyl-7-aminocephalo-sporanic acid, hereinbelow indicated asα-ketoadipyl-7-ACA) and hydrogen peroxide, which chemicallydecarboxylate the α-ketoadipyl-7-ACA to 7β-(4-carboxybutanamido)-ceph-3-em-4-carboxylic acid (orglutaryl-7-aminocephalo-sporanic acid, hereinbelow indicated asGL-7-ACA).

[0006] In a second stage, a specific acylase for GL-7-ACA,glutaryl-7-ACA acylase (E.C. 3.5.1.3), is used, for example that of aPseudomonas type microorganism (U.S. Pat. No. 3,960,662, EP 0496993)over expressed in E. coli, which deacylates the GL-7-ACA into7-amino-ceph-3-em-4-carboxylic acid (or 7-amino cephalosporanic acid,hereinbelow indicated as 7-ACA).

[0007] This two-step enzymatic process for obtaining 7-ACA has been usedon an industrial scale (Conlon et al. Biotechnol. Bioeng. 46, 510,1995).

[0008] Yet another advance in enzymatic processes, is disclosed in EP0846695, in which solid glutaryl-7-ACA is reacted with a heterocyclicgroup that contains at least a nitrogen with or without a sulphur oroxygen atom to produce a 3-modified glutaryl-7-ACA. These 3-derivativesare enzymatically transformed to their corresponding 3-heterocyclicthiomethyl-7-ACA derivatives.

[0009] This procedure can be defined as an enzymatic-chemical-enzymatic(ECE) process, since the isolated GL-7-ACA comes from a bioconversion ofsolubilised cephalosporin C, then GL-7-ACA is reacted with theheterocyclic thiols and finally the 3-heterocyclic thio-derivative isenzymated with GL-7-ACA acylase. The problem with this method is theneed to isolate GL-7-ACA, which given its high water solubility, istechnically difficult and expensive, as described in WO 9535020.

[0010] An additional problem is that the enzyme can only be reused a fewtimes due to the “poisoning” of the biocatalyst by the residualheterocyclic thiols. This poisoning effect is well documented with oneof the thiols used, 5-methyl-1,3,4-thiadiazole-2-thiol (MMTD) (Won etal, App. Biochem. Biotech. 69, 1, 1998).

[0011] The oxidative deamination of the D-adipamido side chain ofcephalosporin C under aerobic conditions into α-ketoadipyl-7-ACA hasbeen described using D-amino acid oxidase (D-AAO) from cell-freeextracts (GB 1,272,769, Glaxo) or in toluene-activated (permeabilised)cells (GB 1,385,685) of the yeast Trigonopsis variabilis or Rhodotorulaglutinis (EP 0517200). In this reaction, molecular oxygen acts as theelectron acceptor and is converted to hydrogen peroxide, whichchemically reacts with the α-ketoadipyl-7-ACA producing itsdecarboxylation into glutaryl-7-ACA. In the presence of large quantitiesof the catalase produced for the above yeasts, the hydrogen peroxide iscleaved to water and molecular oxygen, rendering a mixture ofα-ketoadipyl-7-ACA and glutaryl-7-ACA. The α-ketoadipyl-7-ACA is quiteunstable (GB 1,385,685) and rapidly decomposes to unknown products andhence reduces the yield of glutaryl-7-ACA from 90 to 95% to 60 to 70%,depending on the yeast and strain (Parmar et al, Crit. Rev. Biotechnol.18, 1, 1998; Rietharst, W. and Riechert, A, Chimia 53, 600, 1999). As aresult no industrial application has been described.

[0012] There is therefore a need for an efficient and improved processfor the preparation of 3-thiolated-7-ACA cephalosporanic acidderivatives on an industrial scale. In addition the isolation of stableα-ketoacid derivatives which are important biopharmaceutical compoundswould be beneficial.

STATEMENTS OF INVENTION

[0013] According to the invention there is provided a process forpreparing cephalosporanic acid derivatives comprising the steps of:—

[0014] enzymatically converting a 3-thiolated cephalosporin C compoundof formula III:—

[0015]  into a 3-thiolated-α-ketoadipyl-7-aminocephalosporanic acidderivative of formula IV:

[0016]  wherein R is a heterocyclic group comprising at least a nitrogenatom.

[0017] Preferably the compound of formula III is enzymatically convertedinto a compound of formula IV by an immobilised enzyme system. Mostpreferably the enzyme system comprises co-immobilised D-Amino acidoxidase and catalase.

[0018] Preferably the enzymatic conversion is carried out in thepresence of molecular oxygen, at a pressure of 1 to 5 bar absolute, a pHof from 6.5 to 8.0 and at a temperature of from 15 to 30° C. for aperiod of from 30 mins to 180 mins.

[0019] Preferably the process comprises the step of separating theenzyme system from the reaction mixture, preferably by filtration.

[0020] In one embodiment of the invention the process includes the stepof purifying the compound of formula IV.

[0021] Most preferably the compound is purified using an adsorptioncolumn.

[0022] Preferably the enzymes are co-immobilised using a suitablecross-linker agent in a suitable solid support. The enzymes may be inthe form of crystals of a size suitable for use as a biocatalyst.

[0023] Preferably the enzymatic processes are carried out whilemaintaining the enzyme in dispersion in an aqueous substrate solution.Preferably the or each enzymatic process is carried out in a column.Most preferably the process includes the step of recovering the enzymefor reuse.

[0024] In one embodiment of the invention the compound of formula IV isused without purification in a continuous process for obtaining anyuseful derivative.

[0025] Preferably the R group in compounds of formula III and IV is aheterocyclic group comprising at least one nitrogen atom and optionallya sulphur or oxygen atom.

[0026] Most preferably R is a heterocyclic group selected from any oneor more of the group comprising thienyl, diazolyl, tetrazolyl,thiazolyl, triazinyl, oxazolyl, oxadiazolyl, pyridyl, pirimidinyl, benzothiazolyl, benzimidazolyl, benzoxazolyl, or any derivative thereof,preferably 5-methyl-1,3,4-thiadiazol-2-yl, 1-methyl-1H-tetrazol-5-yl or1,2,5,6-tetrahydro-2-methyl-5,6-dioxo-1,2,4-triazin-3-yl.

[0027] The invention provides a3-thiolated-α-ketoadipyl-7-aminocephalosporanic acid derivative offormula IV whenever prepared by a process of the invention.

[0028] The invention provides a compound of the Formula:—

[0029]  wherein in formula IV, R is1,2,5,6-tetrahydro-2-methyl-5,6-dioxo-1,2,4-triazin-3-yl.

[0030] The invention provides a compound of the Formula:—

[0031]  wherein in formula IV, R is 1-methyl-1H-tetrazol-5-yl.

[0032] The invention also provides use of a compound of formula IV as anintermediate in a process for preparing cephalosporin C antibiotics.

[0033] The invention also provides use of an intermediate compound ofthe formula:—

[0034]  in a process for preparing cephalosporin C antibiotics whereinin formula IV R is 5-methyl-1,3,4-thiadiazol-2-yl.

[0035] The invention further provides a process for preparingcephalosporanic acid derivatives of the invention comprising the stepof:

[0036] enzymatically converting a compound of formula IV to form acompound of formula I

[0037]  wherein R is a heterocyclic group comprising at least onenitrogen atom and R₁ and R₂ are both hydrogen atoms or one of them is ahydrogen atom and the other is an acyl donor.

[0038] Preferably the compound of formula IV is enzymatically convertedto form a compound of formula I using Glutaryl-7-ACA acylase, mostpreferably the enzymation takes place at a temperature of approximately20° C. and at a pH of between 6.5 and 8.0. Preferably the enzyme isimmobilised using a suitable cross-linker agent in a suitable solidsupport.

[0039] Preferably the enzyme is in the form of crystals of a sizesuitable for use as a biocatalyst.

[0040] In one embodiment of the invention the enzymation is carried outwhile maintaining the enzyme in dispersion in an aqueous substratesolution. Preferably the enzymatic process is carried out in a column.Most preferably the process includes the step of recovering the enzymefor reuse.

[0041] The invention also provides use of a compound of formula I as anintermediate in a process for preparing cephalosporin C derivatives.

[0042] The invention further provides a process for preparing3-thiolated cephalosporanic acid derivatives comprising the steps of;—

[0043] enzymatically converting a compound of formula III

[0044]  into a 3-thiolated-α-ketoadipyl-7-aminocephalosporanic acidderivative of formula IV:

[0045] and enzymatically converting a compound of formula IV to form a3-thiolated 7-ACA compound of formula I

[0046] wherein R is a heterocyclic group comprising at least onenitrogen atom and R₁ and R₂ are both hydrogen atoms or one of them is ahydrogen atom and the other is an acyl donor.

[0047] In one embodiment of the invention the compound of formula III isenzymatically converted into a compound of formula I in one step by animmobilised enzyme system. Most preferably the enzyme system comprises acombination of co-immobilised D-amino acid oxidase/catalase in thepresence of immobilised Glutaryl-7-ACA acylase. Preferably theenzymation takes place at a temperature of approximately 20° C. and at apH of between 6.5 and 8.0. Most preferably the enzymes areco-immobilised using a suitable cross-linker agent in a suitable solidsupport.

[0048] Preferably the enzymes are in the form of crystals of a sizesuitable for use as a biocatalyst.

[0049] Most preferably the enzymatic processes are carried out whilemaintaining the enzyme in dispersion in an aqueous substrate solution.

[0050] Preferably the or each enzymatic process is carried out in acolumn. Most preferably the process includes the step of recovering theenzyme for reuse.

[0051] In one embodiment of the invention the compound of formula III isused without purification in a continuous process for obtaining anyuseful derivative.

[0052] The invention also provides a process for preparingcephalosporanic acid derivatives comprising the steps of:—

[0053] reacting cephalosporin C with a thiol compound of the generalformula II

R—SH  II

[0054]  wherein R is a heterocyclic group comprising at least onenitrogen atom,

[0055] to form a 3-thiolated cephalosporin compound of formula III

[0056]  wherein R is as defined above,

[0057] and, after formation of the compound of formula III removingexcess thiol of formula II.

[0058] In one embodiment of the invention the excess thiol is removed byadsorption on an anion exchange resin. Preferably the anion exchangeresin is a microporous resin having a cross-linked acrylic copolymerstructure. Most preferably the anion exchange resin comprises an 8%cross-linking containing functional thialkyl benzyl ammonium group. Theresin may be in the chloride, hydroxy, phosphate or acetate cycle.

[0059] In another embodiment of the invention the excess thiol isremoved by crystallisation. Preferably crystallisation is carried out atan acidic pH.

[0060] In a further embodiment of the invention the excess thiol isremoved by crystallisation followed by adsorption on an anion exchangeresin.

[0061] Preferably the cephalosporin C is in an aqueous medium. Mostpreferably the cephalosporin C is in the form of a concentratedcephalosporin C solution.

[0062] Preferably the reaction is carried out at a pH of between 5.5 and8.0, at a temperature of from 60° C. to 80° C., for a period of from 1to 8 hours. Most preferably the reaction is carried out at a pH ofapproximately 6.0 and at a temperature of approximately 65° C.

[0063] In one embodiment of the invention the thiol compound is presentin an amount of between 1 and 5 mol/mol of cephalosporin C.

[0064] Preferably R is a heterocyclic group comprising at least onenitrogen atom and optionally a sulphur or oxygen atom. Most preferably Ris a heterocyclic group selected from any one or more of thienyl,diazolyl, thiazolyl, tetrazolyl, thiadiazolyl, triazinyl, oxazolyl,oxadiazolyl, pyridyl, pirimidinyl, benzothiazolyl, benzimidazolyl,benzoxazolyl, or any derivative thereof, preferably5-methyl-1,3,4-thiadiazol-2-yl, 1-methyl-tetrazol-5-yl or1,2,5,6-tetrahydro-2-methyl-5,6-dioxo-1,2,4-triazin-3-yl.

[0065] The invention provides a compound of formula III

[0066]  wherein R is a heterocyclic group comprising at least onenitrogen atom,

[0067] The invention provides a compound of the formula:—

[0068]  wherein in formula III R is 5-methyl-1,3,4-thiadiazol-2-yl.

[0069] The invention further provides a compound of the formula:—

[0070]  wherein in formula III R is1,2,5,6-tetrahydro-2-methyl-5,6-dioxo-1,2,4-triazin-3-yl.

[0071] The invention also provides use of a compound of formula III asan intermediate in a process for preparing cephalosporin C derivatives.

[0072] One embodiment of the invention provides a process for preparingcephalosporanic acid derivatives comprising the steps of:—

[0073] enzymatically converting a 3-thiolated cephalosporin C compoundof formula III obtained by a process as described above:—

[0074]  into a 3-thiolated-α-ketoadipyl-7-aminocephalosporanic acidderivative of formula IV:

[0075]  wherein R is a heterocyclic group comprising at least a nitrogenatom.

[0076] In one embodiment of the invention the process additionallycomprises the step of:

[0077] enzymatically converting a 3-thiolated α-ketoadipyl 7-ACAcompound of formula IV

[0078]  to form a 3-thiolated 7-ACA compound of formula I

[0079]  wherein R is a heterocyclic group comprising at least onenitrogen atom and R₁ and R₂ are both hydrogen atoms or one of them is ahydrogen atom and the other is an acyl donor.

[0080] Another embodiment of the invention provides a process forpreparing cephalosporanic acid derivatives comprising the step of:

[0081] enzymatically converting a compound of formula IV

[0082]  to form a compound of formula I

[0083]  wherein R is a heterocyclic group comprising at least onenitrogen atom and R₁ and R₂ are both hydrogen atoms or one of them is ahydrogen atom and the other is an acyl donor.

[0084] Preferably a compound of formula IV is enzymatically converted toform a compound of formula I with Glutaryl-7-ACA acylase.

[0085] Most preferably compounds of formula I, III and IV are in a solidform or in the form of a non-toxic salt thereof.

[0086] The invention provides a process for the preparation ofcephalosporin C antibiotics and derivatives thereof comprising forming acompound of formula III, IV and I as hereinbefore defined and subsequentenzymation of the compound.

[0087] The antibiotic may be any one or more of cefazolin, cefazedone,cefoperazone, cefamandol, cefatriazine, cefotiam and ceftriaxone.

DETAILED DESCRIPTION

[0088] We have found that 3-thiolated cephalosporanic C derivatives maybe enzymated into α-ketoacid derivatives in the presence of aco-immobilised D-amino acid oxidase/catalase system. These α-ketoacidderivatives have been shown to be stable when isolated. A new improvedprocess to obtain 3-thiolated-7-ACA derivatives, both in one step or intwo consecutive enzymatic steps is thereby provided.

[0089] The present invention relates to an improved and more efficientprocess for preparing compounds of formula I from cephalosporin C.

[0090]  wherein R is a heterocyclic group comprising at least onenitrogen atom and R₁ and R₂ are both hydrogen atoms or one of them is ahydrogen atom and the other is an acyl donor.

[0091] The process involves the formation of new stableα-ketoadipyl-7-ACA derivative intermediates. Alternatively the processmay be carried out in a single one pot reaction without the formation ofintermediates.

[0092] It was surprisingly found that the 3-thiolated derivative ofcephalosporin C were very good substrates for the enzymatic reaction byD-amino acid oxidase in the presence of catalase.

[0093] 3-thiolated-cephalosporin C derivatives of formula III

[0094] are prepared from cephalosporin C. The cephalosporin C solutionmay be in a purified or crude form. The cephalosporin C is in the formof any non-toxic salt of cephalosporin C.

[0095] The reaction of nucleophilic substitution in the 3′position iscarried out in an aqueous medium, dissolving the heterocyclic thiol andany non-toxic cephalosporin C salt in water by addition of a basiccompound which form a water soluble salt, such as alkali metalhydroxide, ammonium hydroxide or preferably alkali metal carbonate orbicarbonate. In general, in addition to salts produced as describedabove, any commercially available salt of cephalosporin C and of theheterocyclic thiols can be used in the process of this invention withoutchanging the fundamentals of the process.

[0096] After dissolving the heterocyclic thiol and the cephalosporin C,in separate reaction vessels or jointly, both reactants are mixedtogether in the same reactor, before or after heating the solution to atemperature from about 65° C. to 80° C. at a pH value of between 5.5 and7.0.

[0097] Once the reaction starts, the temperature and pH are maintainedpreferably at approximately 65° C. and 6.0 respectively, for a period oftime of approximately 1 hour to 4 hours.

[0098] The heterocyclic thiol/cephalosporin C molar ratio is animportant variable in the yield of the reaction and has to be optimisedfor each heterocyclic thiol used. Molar ratios are between 1.0 and 4.0,preferably at a molar ratio of approximately 4.

[0099] It was found that at these molar ratios the cephalosporin Cremains quite stable with low β-lactam ring degradation, compared to acephalosporin C solution without the thiol, which is completely degradedwithin 40 min at 80° C.

[0100] Once the cephalosporin C level is below 2% of the initial amount,the reaction mixture is cooled to a temperature from about 2° C. toabout 10° C., with or without acidification at a pH of from pH 3.0 to5.5, preferably approximately 5.2, with strong mineral acids, such ashydrogen halides or oxy acids.

[0101] This acidification step gives in some cases, crystallisation ofthe heterocyclic thiol, with the concomitant possibility of reuse for anew reaction.

[0102] After formation of the compound of formula III excess thiolgroups are selectively removed which allows cephalosporin C derivativesof formula III to be prepared at very high purity levels and with verylow levels (<0.2 mg/ml) of heterocyclic thiols present. A highlyselective removal procedure with the strong anion exchanger AmberliteIRA400 (manufactured by Rohm and Haas) is utilised. This process hasseveral advantages. It allows compounds of Formula III to be used as asubstrate without the poisoning of the enzymes in the next process step.As a result the enzymes may be used repeatedly. In addition the processdoes not require the use of toxic reagents or the need to isolateintermediates thereby providing a continuous process.

[0103] Different resins and types of chromatography may be used on anindustrial scale.

[0104] Several resins were tested grouped in four classes of resinsbased on adsorption, hydrophilic-hydrophobic interaction, cationexchange, and anion exchange. All resins tested based on adsorption(Amberlite XAD-761, Amberlite 7HP, Amberlite 16 HP and Amberlite XAD4)gave similar results, the eluate containing from 22% to 38% of theheterocyclic thiol. The hydrophobic-hydrophilic interaction resinSephadex LH-20 did not retain any thiol (<5%). A similar situation wasfound with the cation exchangers Amberlite® IRC-50, IR-120 and IR-200.However, anion exchangers were found to have the best binding capacityfor heterocyclic thiols ranging from 57-60% in the case of a weak anionexchanger (Amberlite IRA-93).

[0105] It was found that a strong microporous (gel-type I) anion (base)exchange resin Amberlite IRA400 having an 8% cross linking containingfunction trialkyl benzyl ammonium groups gave the highest binding ofheterocyclic thiols (from 92-98%) and low binding of the 3′-positionheterocyclic thiomethyl cephalosporin C derivative (from 2-15%, lessthan 15% for the first cycle and less than 5% for the following cycles).

[0106] Such a microporous resin offers certain advantages. They are lessfragile, require less care in handling and possess higher loadingcapacities. As they have no discrete pores solute ions diffuse throughthe particle to interact with exchange sites. The total exchangecapacity of the mentioned resin is in the order of 1,4 meq/mL.

[0107] It was surprisingly found that Amberlite IRA400 has less bindingcapacity for 3′-heterocyclic thiomethyl derivatives of cephalosporin Cthan for the same derivatives of glutaryl-7-ACA and 7-ACA. In fact the3′-heterocyclic thiomethyl derivative of glutaryl-7-ACA produced withMMTD binds at a level of 76.3% to the column. The same result is foundwith 3′-heterocyclic thiomethyl derivative of 7-ACA, with MMTD, whichbinds at a level of 92.7% to the column. This unexpected behaviour ofAmberlite IRA400 with these three related β-lactam compounds appears toresult from the presence of an ionisable amino group in the 5 positionof the side chain of cephalosporin C compared with glutaryl-7-ACA and7-ACA.

[0108] The removal of heterocyclic thiols by the process of theinvention is particularly advantageous on an industrial scale as theeluate of the column can be used for enzymation without isolation of themodified cephalosporin C and represents a new concept in the field ofcephalosporin intermediates wherein the impurities are bound to thecolumn and the β-lactam derivative is simply eluted by water.

[0109] Once the β-lactam derivative is eluted (less than 5% remainsbound), the column is typically regenerated with a 1.5 N solution of astrong mineral acid, such as hydrogen halide containing variable amountsof an organic solvent, preferably 10-20% acetonitrile. When theconcentration of the thiol in the eluate is higher than 0.2 mg/ml, astrong regeneration using 3 N HCl and 40% acetonitrile may be carriedout. Alternatively regeneration with 1.5M HCl and 1.0 N NaOH is alsopossible.

[0110] After elution of the heterocyclic thiol, the thiol isconcentrated and reused. The column is rinsed with deionised water toremove excess regenerant before the next cycle. The first bed volume ofthe rinse should be performed at the flow rate used for regeneration.The remainder is run at the adsorption flow rate.

[0111] Compounds of formula III are enzymatically converted into newstable α-ketoadipyl-7-ACA derivatives of formula IV by an immobilisedenzyme system.

[0112]  wherein R is a heterocyclic group comprising at least a nitrogenatom with or without a sulphur or oxygen.

[0113] The use of co-immobilised enzymes (D-AAO and catalase) on thesame solid support allows a better hydrogen peroxide removal than withseparate supports. The biocatalyst with both enzymes is easilyrecoverable from the reaction medium and reusable a large number oftimes. This is a necessary and indispensable factor for an industrialprocess.

[0114] A further industrial advantage of the invention is the easytransfer of the chemical solution comprising a compound of formula III,after chromatography in a strong anionic exchange resin (Amberlite®IRA400, manufactured by Rohm and Haas) to an enzymatic reactorcontaining the co-immobilised enzymes. This enables the process to beconducted continuously with a single liquid stream from cephalosporin Cto compound IV.

[0115] The enzymatic stage may be carried out in different ways:

[0116] 1) Chemical reaction without removal of the excess of theheterocyclic thiol and oxidative deamination with immobilised D-AAO.Under these conditions, compound IV is accumulated to about 35 to 40% oftotal β-lactams, which is higher than the 5 to 10% accumulation ofα-ketoadipyl-7-ACA produced when unmodified cephalosporin C is used.Thus indicating the stability of the compound IV.

[0117] 2) The same as 1) including soluble catalase. Under theseconditions, the level of compound IV reaches 70 to 75% of totalβ-lactams in solution with less than 10% of 3-thiolated glutaryl-7-ACA(T′X′G). However the immobilised enzyme and catalase are poisoned withina few cycles by the presence of high levels of heterocyclic thiol (□1mg/mL).

[0118] 3) Chemical reaction with removal of the excess of theheterocyclic thiol by ion exchange chromatography and co-immobilisationof D-AAO and catalase on the same solid support. Under these conditions,compound IV is accumulated from about 80% to 90% of total β-lactams,depending on the pH used. At pH values of approximately 6.5, compound IVis more stable reaching 90% accumulation but the D-AAO is less active(more biocatalyst is needed). At pH values near 7.25, the enzyme is moreactive but compound IV is less stable, obtaining a 80% accumulation. Thepreferred pH is pH 6.75, which provides the lowest loss in D-AAOactivity with good stability of compound IV.

[0119] From the above it is clear that approach 3) is advantageousversus the others but several parameters have to be taken into accountto produce a good biocatalyst with the two enzymes (D-AAO and catalase)co-immobilised:

[0120] a) The source of both enzymes. D-AAO in this invention isobtained from Trigonopsis variabilis CBS 4091 obtained from the Spanishcollection of microorganisms. (CECT, Valencia, Spain). This yeast isgrown under the conditions to induce D-AAO (Kubicek et al, J. Appl.Biochem. 7; 104, 1985) and the enzyme was purified by ammonium sulfatefractionation between 30%-55% as described by Szwjcer et al (Biotechnol.Lett. 7, 1, 1985). Amino acid oxidases may also be sourced fromRodotorula gracilis. Catalase from Micrococcus lisodeikticus is obtainedfrom a commercial source (Fluka, Madrid, Spain), but it may also besourced from Aspergillas niger.

[0121] b) The solid support used. Several carriers are available toimmobilise enzymes. The most popular are: Amberlite® IRA 900 (stronglybasic polystyrene resin with a quaternary amine function), Duolite® A365(weakly basic polystyrene resin with primary functional groups),Duolite® A568 (moderately basic poly-condensed phenolformaldehyderesin), BrCN— activated Sepharose®, vinyl Sepharose® and Eupergit C®(based on a polyacrylic structure and in particular with oxiraneterminal groups). Among Eupergit, two classes are commercialised (RöhmPharma) C and C250L, the latter type is particularly suitable forbinding high molecular weight enzymes, since its contents in oxiranegroups are at least 0.36% compared with the 0.93% in Eupergit C. ThisC250L type show outstanding properties when employed in industrialbiocatalytic processes. The morphology of the carrier, i.e., its narrowparticle size distribution (200 μm) and high mechanical stabilityaccounts for their good properties in stirred-tank reactors. It is notmechanically destroyed in stirred systems and filtration at the end ofthe reaction cycle is quick and very easy to perform. Changes in the pHand ionic strength have no effect on swelling of the matrix. Inaddition, this Eupergit C250L has never been used to immobilise D-aminoacid oxidase and catalase.

[0122] c) The ratio catalase units/D-AAO units. This ratio is normallybigger than 100, but for efficient hydrogen peroxide removal the ratiois preferably about 1500. One unit of D-AAO is defined as the amount ofenzyme that consumes a μmol of O₂ per minute using cephalosporin C assubstrate at pH 8.0 and 25° C. One unit of catalase is defined as theamount of enzyme that decomposes 1 μmol of hydrogen peroxide per minuteat pH 7.0 and 25° C.

[0123] d) The procedure of co-immobilisation. Several immobilisationprotocols can be used. The one chosen in this invention is amodification of the method described by Cramer and Steckham(Tetrahedron, 45, 14645, 1997) for the co-immobilisation ofL-α-glycerolphosphate oxidase with catalase. Typically 100 mg ofEupergit® C250L are suspended in 1.5 ml of coupling buffer (1.0 Mpotassium phosphate buffer pH 8.0) in an Erlenmeyer flask. Then 10-40 Uof D-AAO and 10-20 kU of catalase (Fluka, cat# 60634) are added slowly.The mixture is incubated for 16 h with gentle shaking. After theimmobilisation procedure, the beads are separated by a glass frit andwashed for several times using a 100 mM potassium phosphate buffer pH7.0 at 4° C.

[0124] Once the co-immobilisation is carried out, the enzymaticconversion of the compound III into compound IV is carried out in anaqueous solution of compound III containing from about 0.0016 to 0.004moles and with less than 0.2 mg/ml of the heterocyclic thiol. Thissolution is obtained after passing the solution comprising 3-thiolatedcephalosporin C (compound III) and remaining heterocyclic thiol used inthe nucleophilic displacement of 3 acetoxy group of cephalosporin Cthrough a column of a strong anion exchanger, such as Amberlite® IRA400(Rohm and Hass). The pH of the eluate is adjusted to pH about 6.5 to8.0, preferably to pH 6.75, due to the instability of cephalosporaniccompounds at basic pH values.

[0125] The solution comprising compound III as described above is fedinto a bioreactor, containing wet Eupergit C250L with co-immobilisedD-AAO/catalase, usually D-AAO from 2040 U/g, and catalase, usually from10-30 kU/g. The reaction temperature can be fixed from 15° C. to 35° C.,and is normally fixed at 20° C.

[0126] The molecular oxygen, needed for the oxidative deamination, isblown into solution by a bottom diffuser at a flow rate from 0.01 to 1volume/volume of solution/minute, preferably at 0.1 vvm under a suitablemechanical stirring of about 400 rpm. This bioreactor design ispreferred versus a percolation column containing the immobilised enzymesto avoid the diffusional problems of the molecular oxygen, which reducethe yield of compound IV. The pH is titrated to pH 6.75 by dosing aconcentrated organic or inorganic base, preferably 3 M ammonia, by meansof an autotitrator.

[0127] The conversion is controlled by HPLC and when the conversion ofcompound III is greater than 97%, the reaction is stopped and thesolution filtered off. The time required for such conversion is of theorder 0.5 to 3 hours, depending on the operating conditions, but usuallyapproximately 1 hour.

[0128] Isolation of compound IV, when required, is carried out bydecreasing the pH of the above solution to a pH of about 4.5 to 6.0,preferably 5.0 with the same base used during the enzymatic reaction,and loading into a column packed with the adsorptive resin AmberliteXAD-2. The elution of compound IV is carried out with water at a flowrate of 2-3 bed volumes per hour. Fractions containing the compound IVwith a HPLC purity higher than 90-95% are pooled and lyophilised.

[0129] Compounds of formula IV are subsequently converted into compoundsof formula I by enzymation with glutaryl-7-ACA acylase.

[0130] The process of the invention for preparing compounds of formula Ifrom cephalosporin C may also be carried out in a single one potreaction. In this case filtrate from an anion exchange column comprisingcompounds of formula III is enzymatically converted into compounds offormula I by an immobilised enzyme system comprising D-AAO and catalasein the presence of glutaryl-7-ACA acylase. Compounds of formula I havebeen prepared in this way with a HPLC of approximately 95%. The processis easy and efficient to carry out.

[0131] Using both processes (one-pot or two-steps) of the invention,3-thiolated-7-ACA derivatives are easily and economically prepared.These compounds may by subsequent enzymation with penicillin G acylasefor example, be used for the preparation of semisynthetic β-lactamantibiotics. The β-lactam antibiotics may include any one or more ofcefazolin, cefazedone, cefoperazone, cefamandol, cefatriazine, cefotiamand ceftriaxone.

[0132] The following examples are meant to illustrate the inventionwithout limitation as to its generality.

[0133] Examples 1 to 5 illustrates the preparation of 3-thiolated-7-ACAderivatives of formula III from cephalosporin C.

[0134] Examples 6 to 8 illustrate the enzymatic process for thepreparation of a 3-thiolated α-ketoadipyl-7-ACA derivatives of formulaIV from 3-thiolated cephalosporin C derivatives of formula III.

[0135] Examples 9 to 11 illustrate the enzymatic process for thepreparation of 3-thiolated-7-ACA derivatives (TXA) of formula I from3-thiolated derivatives of formula III via the formation of stableα-ketoadipyl-7-ACA derivatives of formula IV.

[0136] Examples 12 to 14 illustrate the enzymatic process for thepreparation of 3-thiolated-7-ACA derivatives of formula I from3-thiolated derivatives of formula III in a single step (one pot).

EXAMPLE 1 Preparation of7-β-(5-amino-5-carboxypentanamido)-3-(5-methyl-1,3,4-thiadiazole-2-ylthiomethyl)-3-cephem-4-carboxylic acid (TDC).

[0137] To a glass-lined reactor containing 600 mL of deionised water,31.73 g (0.24 moles) of 2-mercapto-5-methyl-1,3,4-thiadiazole (MMTD)were added and the reactor was heated with stirring to a temperature ofabout 65° C. The pH of the mixture was adjusted to pH of about 6.0 bythe addition of about 10 g of sodium carbonate.

[0138] In a separate glass-lined flask, a solution of concentratedsodium cephalosporin C (98% purity by HPLC) was prepared by dissolving33.23 g of sodium cephalosporin C (75% free acid, 0.06 moles) in 200 mlof water. When the MMTD was dissolved, the concentrated cephalosporin Csolution was added and the mixture was stirred at approximately 65° C.for 240 minutes, controlling the reaction kinetic until the level ofcephalosporin C was below 2%. The following reaction kinetics werefound: Cephalosporin C Time (min) (moles) TDC (moles) MMTD (moles) 00.06 0.00 0.24 120 0.01 0.039 0.20 240 0.0012 0.042 0.195

[0139] The reaction mixture was then cooled to about 4° C., where thecrystallisation of the excess of MMTD begins. The pH was acidified withstirring (150 rpm) to a pH 5.2 with 37% hydrochloride acid and leftunder slow stirring (50 rpm) for 60 minutes for the completion ofcrystallisation.

[0140] The precipitated MMTD was filtered and dried at 35° C. undervacuum. 23 g of recovered MMTD was obtained (purity 99% by HPLC) with arecovery yield of about 95%.

[0141] The filtrate (825 ml) containing 0.042 moles of the TDC and MMTD0.016 moles was adjusted to pH 7.25° with 3 M ammonia and loaded onto anAmberlite IRA-400 column in chloride cycle (bed volume equal to 180 ml)covered with deionised water at flow rate 20 ml/min. Once loaded, thecolumn was washed with deionised water (ca 100 ml) until 97% recovery ofloaded TDC with a 94% purity by HPLC. The pH of the effluent was about5.4 and was neutralised to 7.0 with 3 M ammonia. The remaining MMTD was0.0009 moles (<0.2 mg/ml), which is less than 6% of the remaining MMTDafter its crystallisation by decreasing the pH. With this low level ofMMTD (<1% of the original MMTD after chemical reaction), enzymation ofTDC is possible.

[0142] Typically the column is regenerated with 1 L of 1.5 M HClcontaining 10% acetonitrile and rinsed free of the excess regenerationby washing with 2 litres of deionised water. When required (MMTD>0.2mg/ml) the resin can be subjected to a strong regeneration using 1 litreof 3 M HCl with 40% acetonitrile. Alternatively, regeneration with 1.5 MHCl and 1.0 N NaOH is also possible.

[0143] To further characterise the TDC solution at pH 5.0, it was loadedonto a Amberlite XAD-2 adsorption column and the column was washed withwater. After washing, the resin was eluted with water, and 25 mlportions were pooled. A fraction containing 98.5% TDC by HPLC waslyophylised and subjected to analysis:

[0144] Elemental Analysis for the product C₁₇H₂₀N₅O₆S₃. 2H₂O (TDC),calculated C 37.42; H 4.43; N 12.84; S 17.63; found 37.27; H 4.3; N13.11; S 17.51.

[0145]¹H-NMR (DMSO/DCl) (δ ppm): 1.57 (m, 2H, —CH₂—); 1.63 (m, 2H,—CH₂—); 2.18 (m, 2H, —CH₂—); 2.64 (s, 3H, CH₃); 3.55, 373(J=18 Hz, 2H,—CH2—); 3.83 (t, 1H, —CH—); 4.18-4.46 (d, J=13 Hz, 2H, —CH₂—); 5.02 (d,J=3 Hz, 1H, C-6); 5.6 (d, J=3 Hz, 1H, C-7).

EXAMPLE 2 Comparative Example: Preparation of7-β-(5-amino-5-carboxypentanamido)-3-(5-methyl-1,3,4-thiadiazole-2-ylthiomethyl)-3-cephem-4-carboxylicacid on different columns.

[0146] The TDC derivative was prepared as described in Example 1 and thefiltrate containing it was loaded onto different types of resins.

[0147] The following data was obtained after washing with 100 ml ofwater in the first cycle of column usage: Eluted TDC Eluted Resin Type(%) MMTD (%) Amberlite IRA-400 Strong anion 86 2 exchanger Diaion SA10AStrong anion 67 15 exchanger Amberlite IRA93 Weak anion exchanger 87 45Amberlite IRC-50 Weak cation exchanger 93 92 Amberlite IRC-200 Strongcation 73 98 exchanger Amberlite XAD-761 Adsorption 86 22 AmberliteXAD-7 HP Adsorption 77 23 Amberlite XAD-16 HP Adsorption 75 35 AmberliteXAD-4 Adsorption 68 25 Amberlite XAD-1180 Adsorption 78 38 SephadexLH-20 Hydrophobic 98.5 95 hydrophilic

[0148] Amberlite IRA-400 gave the best results. A high elution of TDCwas observed with low elution of MMTD. Using other anion exchangecolumns higher amounts of MMTD were also eluted. The other anionexchangers showed a high dual binding of thiol and TDC.

EXAMPLE 3 Specificity of TDC for Amberlite IRA-400

[0149] The glutaryl-7-ACA derivative (TDG) and 7-ACA derivative (7-TDA)were prepared as in Example 1 using glutaryl-7-ACA and 7-ACA as startingmaterial. The following data was obtained from the filtrate of theAmberlite IRA400. Compound in TDG or TDA Resin the elute eluted (%) MMTDeluted (%) Amberlite IRA- TDG 23.7 3.0 400 TDA* 7.3 1.4

[0150] In contrast to TDC, both TDG and TDA appear to remain bound tothe Amberlite IRA-400 as well as the MMTD.

EXAMPLE 4 Preparation of7-β-(5-amino-5-carboxypentanamido)-3-[(1-methyl-1H-tetrazol-5-yl)-thiomethyl]-cephalosporanicacid (TZC).

[0151] To a glass-lined reactor containing 600 mL of deionised water28.16 g (0.24 moles) of 5-mercapto-1-methyltetrazole (MMTZ) were addedand the reactor was heated with stirring to a temperature of about 70°C. The pH of the mixture was adjusted to a pH of about 5.7-5.8 by theaddition of about 12 g of sodium carbonate.

[0152] In a separate glass-lined flask, a solution of concentratedsodium cephalosporin C was prepared by dissolving 33.23 g of sodiumcephalosporin C (75% free acid, 0.06 moles purity 98% by HPLC) in 200 mlof water. When the MMTZ was dissolved, the concentrated cephalosporin Csolution was added and the mixture was stirred at about 70° C. for 120minutes, controlling the reaction kinetic until the level ofcephalosporin C was below 3%. Cephalosporin C Time (min) (moles) TZC(moles) MMTZ (moles) 0 0.06 0 0.24 120 0.0017 0.040 0.19

[0153] The reaction mixture was cooled at about 4° C., butcrystallisation of the excess of MMTZ did not start, even when the pHwas decreased. The solution containing 0.04 moles of the TZC derivativefrom MMTZ and 0.19 moles of MMTZ was adjusted to pH 7.25 with 3 Mammonia and loaded onto an Amberlite IRA-400 column in chloride cycle(bed volume equal to 150 ml) covered with deionised water at flow rate20 ml/min. After the first pass through the column the remaining MMTZwas higher than 13% of the initial (0.032 moles).

[0154] For this reason, the eluate was loaded onto another AmberliteIRA-400 (bed volume equal to 60 ml) column under the same conditions asdescribed above to decrease the level of MMTZ.

[0155] Once loaded, the column was washed with deionised water (ca 90ml) until 97% recovery of loaded TZC with a 87% purity by HPLC. The pHof the effluent was about 5.4 and was neutralised to pH 7.0 with 3 Mammonia. The remaining MMTZ concentration was 0.0013 moles, which isless than 1% of the original MMTZ after chemical reaction. With this lowlevel of MMTZ, enzymation of the derivative is possible withoutpoisoning the enzyme.

[0156] The columns were regenerated with 1 L of 1.5 M HCl containing 10%acetonitrile and rinsed free of the excess regeneration by washing with2 litres of deionised water. Alternatively, regeneration with 1.5 M HCland 1.0 N NaOH is also possible.

EXAMPLE 5 Preparation of7-β-(5-amino-5-carboxypentanamido)-3-[(1,2,5,6-terahydro-2-methyl-5,6-dioxo-1,2,4-triazin-3-yl)-thiomethyl]-cephalosporanicacid (TTC).

[0157] To a glass-lined reactor containing 600 ml of deionised water37.96 g (0.24 moles) of2,5-dihydro-3-mercapto-2-methyl-5,6-dioxo-1,2,4-triazine (here belowindicated as TTZ) were added and the reactor was heated with stirring toa temperature of approximately 75° C. The pH of the mixture was adjustedto pH of approximately 6.7 by the addition of approximately 12 g ofsodium carbonate.

[0158] In a separate glass-lined flask, a solution of concentratedsodium cephalosporin C was prepared by dissolving 33.23 g of sodiumcephalosporin C (75% free acid, 0.06 moles, purity 98% by HPLC) in 200ml of water. When the TTZ was dissolved, the concentrated cephalosporinC solution was added and the mixture was stirred at approximately 75° C.for 75 minutes, controlling the reaction kinetic until the level ofcephalosporin C was below 2%. Cephalosporin C Time (min) (moles) TTC(moles) TTZ (moles) 0 0.06 0 0.24 75 0.0011 0.036 0.19

[0159] The reaction mixture was cooled at approximately 4° C., butcrystallisation of the excess of TTZ does not start, even when the pHwas decreased. The solution containing the 0.036 moles of TTC and 0.19moles of TTZ was adjusted to pH 7.25 with 3 M ammonia and loaded onto anAmberlite IRA-400 column in chloride cycle (bed volume equal to 209 ml)covered with deionised water at flow rate 20 ml/min. After the firstcolumn the remaining TTZ was 0.015 moles.

[0160] For this reason, the eluate was loaded onto another AmberliteIRA400 column (with the same bed volume) under the same conditions asdescribed above to decrease the level of TTZ.

[0161] Once loaded, the column was washed with deionised water (ca 120ml) until 60% recovery of loaded TTC with a 90% purity by HPLC. The pHof the effluent was about 5.4 and was neutralised to pH 7.0 with 3 Mammonia. The remaining TTZ concentration was 0.00096 moles, which isless than 1% of the original TTZ after chemical reaction. With thislevel of TTZ, enzymation of the derivative is possible without poisoningthe enzyme.

[0162] The columns were regenerated with 1 L of 1.5 M HCl containing 10%acetonitrile and rinsed free of the excess regeneration by washing with2 litres of deionised water. Alternatively, regeneration with 1.5 M HCland 1.0 N NaOH is also possible.

EXAMPLE 6 Preparation of7β-(5-carboxy-5-oxopentamide)-3-[(5-methyl-1,3,4-thiadiazole-2-yl)-thiomethyl]cephalosporanic acid (TDK).

[0163] A filtrate (80 ml) from the strong anion exchanger Amberlite®IRA-400 containing 0.0035 moles of7β-(5-amino-5-carboxypentamido)-3-[(5-methyl-1,3,4-thiadiazole-2-yl)-thiomethyl]cephalosporanic acid (TDC) with 94.3% purity (HPLC) and less than 0.2mg/mL of 2-mercapto-5-methyl-1,3,4-thiadiazole (MMTD) was adjusted to pH6.75 with 3 M ammonia.

[0164] The TDC solution was fed into a 0.125 litre stirred glass vesselwith 30.76 g of wet Eupergit C250L with a co-immobilised D-amino acidoxidase/catalase system (11.77 U of DAAO/g and 15 kU of catalase/g).

[0165] The conversion was performed at 20° C., 400 rpm and with anoxygen flow through a bottom diffuser of 0.1 vol/vol/min at 1 barabsolute pressure. The pH was titrated to pH 6.75 with 3 M ammonia by anautotitrator.

[0166] The conversion was controlled by HPLC in a reverse phase columnNucleosil 120 3-C18 125×8×4 mm. The mobile phase was 20 mM acetateammonium pH 5.5 containing 4% acetonitrile at 1 ml/min with a 260 nmdetection. The TDC appeared at 7.0 minutes, the TDK at 8.5 min and the3-thiolated glutaryl-7-ACA intermediate (TDG) at 11.5 min.

[0167] Representative samples of the reaction mixture except for theenzyme were taken and the results obtained are given as percentage oftotal β-lactams in the following table: TDC TDK TDG side products Time(min.) [%] [%] [%] [%] 0 94.3 0.0 0.0 5.7 15 68.3 25.9 0.0 5.8 60 2.689.9 0.9 6.6

[0168] When the % of remaining TDC was less than 3%, the reaction wasstopped and the reaction solution was filtered off.

[0169] To isolate TDK, the resulting solution was adjusted to pH 5.0with 3 M ammonia and passed through a column packed with 40 g of theadsorptive resin Amberlite XAD-2 (68.7 ml of bed volume). Elution wascarried out with water at a flow rate of 200 ml/h (about 3 bed volumesper hour). Fractions of 25 ml containing TDK with a purity>95% (HPLC)were pooled and lyophilised to obtain the target product as a solid tofurther analyse it. After the elution process, the adsorbent surface isreactivated by applying 2 bed volumes of regeneration solution (25%methanol in water at 3 bed volumes per hour). Before the column can beused again, this solution is removed from the column. Afterequilibration with water in excess (about 15 bed volumes), the column isready for re-use.

[0170]¹H-NMR (DMSO) (δ ppm): 1.66 (m, 2H, —CH₂—); 2.16 (t, 2H, —CH₂—);2.50 (t, 2H, —CH₂—); 2.65 (s, 3H, —CH3—); 3.34-3.58 (J=17.4 Hz, 2H,—CH2—); 4.31-4.48 (J=12.1 Hz, 2H, —CH₂—); 4.94 (J=5.1 Hz, 1H, C-6); 5.5(d,d, J=5.1 Hz, J=8.4 Hz, 1H, C-7).

EXAMPLE 7 Preparation of7β-(5carboxy-5-oxopentanamide)-3-[(1-methyl-1H-tetrazol-5-yl)-thiomethyl]-cephalosporanicacid (TZK).

[0171] A filtrate (100 ml) from the strong anion exchanger Amberlite®IRA-400 containing 0.0039 moles of7-(5′-amidoadipamido)-3-[(1-methyl-1H-tetrazol-5-yl)-thiomethyl]-cephalosporanicacid (TZC) with 90.1% purity (HPLC) and less than 0.2 mg/ml of5-mercapto-1-methyltetrazole (MMTZ) was adjusted to pH 6.75 with 3 Mammonia.

[0172] The TZC solution was fed into a 0.125 litre stirred glass vesselwith 30.76 g of wet Eupergit C250L with a coimmobilised D-amino acidoxidase/catalase system (11.77 U of DAAO/g and 15 kU of catalase/g).

[0173] The conversion was performed at 20° C., 400 rpm and with anoxygen flow through a bottom diffuser of 0.1 vol/vol/min at 1 barabsolute pressure. The pH was titrated to pH 6.75 with 3 M ammonia by anautotitrator.

[0174] The conversion was controlled by HPLC on a reverse phase columnNucleosil 120 3-C18 125×8×4 mm. The mobile phase was 20 mM ammoniumacetate pH 5.5 containing 4% acetonitrile at 1 ml/min with a 260 nmdetection. The TZC appeared at 3.0 minutes, the TZK at 3.6 min and the3-thiolated glutaryl-7-ACA intermediate (TZG) at 4.6 min.

[0175] Representative samples of the reaction mixture except for theenzyme were taken and the results obtained are given as percentage oftotal β-lactams in the following table: TZC TZK TZG Side products Time(min.) [%] [%] [%] [%] 0 90.1 0.0 0.0 9.9 15 31.3 57.8 0.8 10.1 45 0.487.3 1.6 10.7

[0176] When the % of remaining TZC was less than 3%, the reaction wasstopped and the reaction solution was filtered off.

[0177] To isolate the TZK, the resulting solution was adjusted to pH 5.0with 3 M ammonia and passed through a column packed with 40 g of theadsorptive resin Amberlite XAD-2 (68.7 ml of bed volume). Elution wascarried out with water at a flow rate of 200 ml/h (about 3 bed volumesper hour). Fractions of 25 ml containing the TZK with a purity ≧93%(HPLC) were pooled and then lyophilised to obtain the target product asa solid to further analyse it. After the elution process, the adsorbentsurface is reactivated applying 2 bed volumes of regeneration solution(25% methanol in water at 3 bed volumes per hour). Before the column canbe used again, this solution is removed from the column. Afterequilibration with water in excess (about 15 bed volumes), the column isready for re-use.

[0178]¹H-NMR (DMSO) (δ ppm): 1.61 (m, 2H, —CH₂—); 2.12 (t, 2H, —CH2—);2.45 (t, 2H, —CH₂—); 3.33-3.56 (J=17.4 Hz, 2H, —CH₂—); 3.86 (s, 3H,—CH3—); 4.18-4.35 J=12.6 Hz, 2H, —CH₂—); 4.88 (d, J=4.8 Hz, 1H, C-6);5.46 (d,d, J=4.8 Hz, J=8.2 Hz, 1H, C-7).

EXAMPLE 8 Preparation of7β-(5carboxy-5-oxopentanamide)-3-[(1,2,5,6-tetrahydro-2-methyl-5,6-dioxo-1,2,4-triazin-3-yl)-thiomethyl]-cephalosporanicacid (TTK).

[0179] A filtrate (50 ml) from the strong anion exchanger Amberlite®IRA-400 containing 0.0016 moles of7β-(5-amino5-carboxypentamido)-3-[(1,2,5,6-terahydro-2-methyl-5,6-dioxo-1,2,4-triazin-3-yl)-thiomethyl]-cephalosporanicacid (TTC) with 89.86% purity (HPLC) and less than 0.2 mg/ml of2,5-dihydro-3-mercapto-2-methyl-5,6-dioxo-1,2,4-triazine (TTZ) wasadjusted to pH 6.75 with 3 M ammonia.

[0180] The TTC solution was fed into a 0.125 litre stirred glass vesselwith 30.76 g of wet Eupergit C250L with a co-immobilised D-amino acidoxidase/catalase system (11.77 U of DAAO/g and 15 kU of catalase/g).

[0181] The conversion was performed at 20° C., 400 rpm and with anoxygen flow through a bottom diffuser of 0.1 vol/vol/min at 1 barabsolute pressure. The pH was titrated to pH 6.75 with 3 M ammonia by anautotitrator.

[0182] The conversion was controlled by HPLC in a column Eclipse® XDB-C85μm 4.6×150 mm. The mobile phase was 35% methanol in 10 mM TBHS(tetrabutylammonium hydrogen sulfate) and 15 mM potassium dihydrogenphosphate at 1 ml/min with 260 nm. The TTC appeared at 2.6 minutes, the3-thiolated glutaryl-7-ACA intermediate (TfG) at 5.5 min and the TTK at6.6 min.

[0183] Representative samples of the reaction mixture except for theenzyme were taken and the results obtained are given as percentage oftotal β-lactams in the following table: TTC TTK TTG Side products Time(min) [%] [%] [%] [%] 0 89.86 0.00 0.00 10.14 30 17.26 71.37 0.89 10.4860 0.55 86.52 1.88 11.05

[0184] When the % of remaining TTC was less than 3%, the reaction wasstopped and the reaction solution was filtered off.

[0185] To isolate the TTK, the resulting solution was adjusted to pH 5.0with 3 M ammonia and passed through a column packed with 40 g of theadsorptive resin Amberlite XAD-2 (68.7 ml of bed volume). Elution wascarried out with water at a flow rate of 200 ml/h (about 3 bed volumesper hour). Fractions of 25 ml containing the TTK with a purity>90%(HPLC) were pooled and then lyophilised to obtain the target product assolid to further analyse it. After the elution process, the adsorbentsurface is reactivated by applying 2 bed volumes of regenerationsolution (25% methanol in water at 3 bed volumes per hour). Before thecolumn can be used again, this solution is removed from the column.After equilibration with water in excess (about 15 bed volumes), thecolumn is ready for re-use.

[0186]¹H-NMR (D₂O) (6 ppm): 1.89 (m, 2H, —CH₂—); 2.38 (t, 2H, —CH₂—);2.81 (t, 2H, —CH₂—); 3.45-3.72 J=17.7 Hz, 2H, —CH₂—); 3.65 (s, 3H,—CH3—); 4.054.34 U=13.2 Hz, 2H, —CH₂—); 5.10 (d, J=4.7 Hz, 1H, C-6);5.62 (d, J=4.7 Hz, 1H, C-7).

EXAMPLE 9 Synthesis of7-amino-3-[(5-methyl-1,3,4-thiadiazol-2-yl)-thiomethyl]-cephalosporanicacid (TDA).

[0187] A filtrate (50 ml) from the strong anion exchanger Amberlite®(IRA-400 containing 0.0011 moles of7β-(5-amino-5-carboxypentamido)-3-[(5-methyl-1,3,4-thiadiazole-2-yl)-thiomethyl]cephalosporanic acid (here below indicated as TDC) with 94.01% purity(HPLC) and less than 0.2 mg/ml of 2-mercapto-5-methyl-1,3,4-thiadiazole(MMTD) was adjusted to pH 6.75 with 3 M ammonia.

[0188] The TDC solution was fed into a 0.125 litre stirred glass vesselwith 16 g of wet Eupergit C250L with a co-immobilised D-amino acidoxidase/catalase system (25 U of DAAO/g and 30 kU of catalase/g).

[0189] The conversion was performed at 20° C., 400 rpm and with anoxygen flow through a bottom diffuser of 0.1 vol/vol/min at 1 barabsolute pressure. The pH was titrated to pH 6.75 with 3 M ammonia by anautotitrator.

[0190] The conversion was controlled by HPLC in a reverse phase columnEclipse XDB-C8 150 mm×4.6 mm ID×5μm; the mobile phase was 10 mMtetrabutylammonium hydrogen sulfate, 15 mM potassium dihydrogenphosphate, pH 6.5 containing 35% methanol at 1 ml/min with a 260 nmdetection. The TDC appeared at 3.0 min, the TDK at 10.9 min and the TDGat 8.1 min, respectively.

[0191] Representative samples of the reaction mixture except for theenzyme were taken and the result obtained are reported as percentage oftotal β-lactams in the following table: Time (min) % TDC % TDK % TDG %Side products 0 94.57 0.00 0.00 9.43 15 53.86 36.19 1.66 8.29 60 0.0088.27 5.48 6.25

[0192] When the % of remaining TDC was smaller than 3%, the reaction wasstopped and the reaction solution, containing TDK with a HPLC purity of88.27%, was filtered off, and adjusted to pH 7.25 with 3 M ammonia.

[0193] The TDK solution was fed into a 0.125 litre stirred glass vesselwith 23 g of wet Glutaryl-7-ACA Acylase (87 U/g). The conversion wasperformed at 20° C., 400 rpm at 1 bar absolute pressure. The pH wastitrated to pH 7.25 with 3 M ammonia by an autotitrator.

[0194] The conversion was controlled by HPLC under the above-mentionedconditions. The TDA appeared at 4.1 minutes. Representative samples ofthe reaction mixture except for the enzyme were taken and the resultobtained are reported as percentage of total β-lactams in the followingtable: Time (min) % TDK % TDG % TDA % Side products 0 88.27 5.48 0.06.25 30 11.82 3.50 73.76 10.92 60 2.6 0.0 88.59 8.81

[0195] When the % of remaining TDK was smaller than 3%, the reaction wasstopped. The reaction solution contained TDA with a HPLC purity of88.59%.

EXAMPLE 10 Synthesis of7-amino-3-[(1-methyl-1H-tetrazol-5-yl)-thiomethyl]-cephalosporanic acid(TZA).

[0196] A filtrate (50 mL) from the strong anion exchanger Amberlite®IRA-400 containing 0.00195 moles of7β-(5-amino-5-carboxypentamido)-3-[(1-methyl-1H-tetrazol-5-yl)-thiomethyl]cephalosporanic acid (TZC) with 91.74% purity (HPLC) and less than 0.2mg/ml of 5-mercapto-1-methyltetrazole (MMTZ) was adjusted to pH 6.75with 3 M ammonia.

[0197] The TZC solution was fed into a 0.125 litre stirred glass vesselwith 16 g of wet Eupergit C250L with a coimmobilised D-amino acidoxidase/catalase system (25 U of DAAO/g and 30 kU of catalase/g).

[0198] The conversion was performed at 20° C., 400 rpm and with anoxygen flow through a bottom diffuser of 0.1 vol/vol/min at 1 barabsolute pressure. The pH was titrated to pH 6.75 with 3 M ammonia by anautotitrator.

[0199] The conversion was controlled by HPLC in a reverse phase columnEclipse XDB-C8 150 mm×4.6 mm ID×5μm; the mobile phase was 10 mMtetrabutylammonium hydrogen sulfate, 15 mM potassium dihydrogenphosphate, pH 6.5 containing 35% methanol at 1 ml/min with a 260 nmdetection. The TZC appeared at 2.1 minutes, the TZK at 5.0 min and theTZG at 4.3 min, respectively.

[0200] Representative samples of the reaction mixture except for theenzyme were taken and the result obtained are reported as percentage oftotal β-lactams in the following table: Time (min) % TZC % TZK % TZG %Side products 0 91.74 0.00 0.00 8.26 15 26.23 58.78 5.13 9.86 45 0.5982.42 5.34 11.65

[0201] When the % of remaining TZC was smaller than 3%, the reaction wasstopped and the reaction solution, containing TZK with a HPLC purity of82.42%, was filtered off, and adjusted to pH 7.25 with 3 M ammonia.

[0202] The TZK solution was fed into a 0.125 litre stirred glass vesselwith 23 g of wet Glutaryl-7-ACA Acylase (87 U/g). The conversion wasperformed at 20° C., 400 rpm at 1 bar absolute pressure. The pH wastitrated to pH 7.25 with 3 M ammonia by an autotitrator.

[0203] The conversion was controlled by HPLC under the above-mentionedconditions. The TZA appeared at 2.5 min. Representative samples of thereaction mixture except for the enzyme were taken and the resultobtained are reported as percentage of total β-lactams in the followingtable: Time (min) % TZK % TZG % TZA % Side products 0 82.42 5.34 0.0012.24 15 41.52 5.05 39.26 14.17 150 12.98 3.63 67.02 16.37 300 2.60 0.6280.77 16.01

[0204] When the % of remaining TZK was smaller than 3%, the reaction wasstopped. The reaction solution contained TZA with a HPLC purity of80.77.

EXAMPLE 11 Synthesis of7-amino-3-[(1,2,5,6-tetrahydro-2-methyl-5,6-dioxo-1,2,4-triazin-3-yl)-thiomethyl]-cephalosporanicacid (TTA).

[0205] A filtrate (50 mL) from the strong anion exchanger Amberlite 0IRA-400 containing 0.0014 moles of7β-(5-amino-5-carboxypentamido)-3-[(1,2,5,6-tetrahydro-2-methyl-5,6-dioxo-1,2,4-triazin-3-yl)-thiomethyl]cephalosporanicacid (hereinbelow indicated as TTC) with 92.2% purity (HPLC) and lessthan 0.2 mg/ml of2,5-dihydro-3-mercapto-2-methyl-5,6-dioxo-1,2,4-triazine (TTZ) wasadjusted to pH 6.75 with 3 M ammonia.

[0206] The TTC solution was fed into a 0.125 litre stirred glass vesselwith 16 g of wet Eupergit C250L with a coimmobilized D-amino acidoxidase/catalase system (25 U of DAAO/g and 30 kU of catalase/g).

[0207] The conversion was performed at 20° C., 400 rpm and with anoxygen flow through a bottom diffuser of 0.1 vol/vol/min at 1 barabsolute pressure. The pH was titrated to pH 6.75 with 3 M ammonia by anautotitrator.

[0208] The conversion was controlled by HPLC in a reverse phase columnEclipse XDB-C8 150 mm×4.6 mm ID×5μm; the mobile phase was 10 mMtetrabutylammonium hydrogen sulfate, 15 mM potassium dihydrogenphosphate, pH 6.5 containing 35% methanol at 1 ml/min with a 260 nmdetection. The TTC appeared at 2.4 minutes, the TTK at 6.1 min and theTTG at 5.5 min, respectively.

[0209] Representative samples of the reaction mixture except for theenzyme were taken and the result obtained are reported as percentage oftotal β-lactams in the following table: Time (min) % TTC % TTK % TTG %Side products 0 92.29 0.00 0.00 7.71 15 32.10 60.97 0.10 6.83 60 1.2290.00 0.12 8.66

[0210] When the % of remaining TTC was smaller than 3%, the reaction wasstopped and the reaction solution, containing TTK with a HPLC purity of90.0%, was filtered off, and adjusted to pH 7.25 with 3 M ammonia.

[0211] The TTK solution was fed into a 0.125 litre stirred glass vesselwith 23 g of wet Glutaryl-7-ACA Acylase (87 U/g). The conversion wasperformed at 20° C., 400 rpm at 1 bar absolute pressure. The pH wastitrated to pH 7.25 with 3 M ammonia by an autotitrator.

[0212] The conversion was controlled by HPLC under the above-mentionedconditions. The TTA appeared at 2.9 min. Representative samples of thereaction mixture except for the enzyme were taken and the resultobtained are reported as percentage of total β-lactams in the followingtable: Time (min) % TTK % TTG % TTA % Side products 0 90.00 0.12 0.009.88 15 24.12 0.00 62.94 12.94 60 1.23 0.00 78.40 20.37

[0213] When the % of remaining TTK was smaller than 3%, the reaction wasstopped. The reaction solution contained TTA with a HPLC purity of78.40%.

EXAMPLE 12: Synthesis of7-amino-3-[(5-methyl-1,3,4-thiadiazol-2-yl)-thiomethyl]-cephalosporanicacid (TDA) in a single step

[0214] A filtrate (50 ml) from the strong anion exchanger Amberlite®IRA-400 containing 0.0011 moles of7β-(5-amino-5-carboxypentamido)-3-[(5-methyl-1,3,4-thiadiazole-2-yl)-thiomethyl]cephalosporanic acid (hereinbelow indicated as TDC) with 95.41% purity(HPLC) and less than 0.2 mg/ml of 2-mercapto-5-methyl-1,3,4-thiadiazole(MMTD) was adjusted to pH 7.25 with 3 M ammonia.

[0215] The TDC solution was fed into a 0.125 litre stirred glass vesselwith 16 g of wet Eupergit C250L with a co-immobilised D-amino acidoxidase/catalase system (25 U of DAAO/g and 30 kU of catalase/g) and 23g of wet Glutaryl-7-ACA Acylase (87 U/g).

[0216] The conversion was performed at 20° C., 400 rpm and with anoxygen flow through a bottom diffuser of 0.1 vol/vol/min at 1 barabsolute pressure. The pH was titrated to pH 7.25 with 3 M ammonia by anautotitrator.

[0217] The conversion was controlled by HPLC in a reverse phase columnEclipse XDB-C8 150 mm×4.6 mm ID×5μ; the mobile phase was 10 mMtetrabutylammonium hydrogen sulfate, 15 mM potassium dihydrogenphosphate, pH 6.5 containing 35% methanol at 1 ml/min with a 260 nmdetection. The TDC appeared at 3.0 min, the TDK at 10.9 min, the TDG at8.1 min and the TDA at 4.1 min, respectively.

[0218] Representative samples of the reaction mixture except for theenzyme were taken and the result obtained are reported as percentage oftotal β-lactams in the following table: Time (min) % TDC % TDK % TDG %TDA % Side Products 0 95.42 0.00 0.00 0.00 4.58 15 28.50 27.11 3.4336.51 4.45 60 13.27 11.87 0.00 70.17 4.69 150 0.00 0.00 0.00 95.13 4.87

[0219] When the % of remaining TDK was smaller than 3%, the reaction wasstopped. The reaction solution contained TDA with a HPLC purity of95.13%.

EXAMPLE 13 Synthesis of7-amino-3-[(1-methyl-1H-tetrazol-5-yl)-thiomethyl]-cephalosporanic acid(TZA) in a single step.

[0220] A filtrate (50 ml) from the strong anion exchanger Amberlite®IRA-400 containing 0.00195 moles of7β-(5-amino-5-carboxypentamido)-3-[(1-methyl-1H-tetrazol-5-yl)-thiomethyl]cephalosporanic acid (hereinbelow indicated as TZC) with 93.55% purity(HPLC) and less than 0.2 mg/ml of 5-mercapto-1-methyltetrazole (MMTZ)was adjusted to pH 7.25 with 3 M ammonia.

[0221] The TZC solution was fed into a 0.125 litre stirred glass vesselwith 16 g of wet Eupergit C250L with a co-immobilised D-amino acidoxidase/catalase system (25 U of DAAO/g and 30 kU of catalase/g) and 23g of wet Glutaryl-7-ACA Acylase (87 U/g).

[0222] The conversion was performed at 20° C., 400 rpm and with anoxygen flow through a bottom diffuser of 0.1 vol/vol/min at 1 barabsolute pressure. The pH was titrated to pH 7.25 with 3 M ammonia by anautotitrator.

[0223] The conversion was controlled by HPLC in a reverse phase columnEclipse XDB-C8 150 mm×4.6 mm ID×5μ; the mobile phase was 10 mMtetrabutylammonium hydrogen sulfate, 15 mM potassium dihydrogenphosphate, pH 6.5 containing 35% methanol at 1 ml/min with a 260 nmdetection. The TZC appeared at 2.1 minutes, the TZK at 5.0 min, the TZGat 4.3 min, and the TZA at 2.5 min, respectively.

[0224] Representative samples of the reaction mixture except for theenzyme were taken and the result obtained are reported as percentage oftotal β-lactams in the following table: Time (min) % TZC % TZK % TZG %TZA % Side products 0 93.55 0.00 0.00 0.00 6.45 30 11.46 53.00 4.7819.39 11.37 90 0.00 40.20 7.32 36.56 15.92 180 0.00 25.69 5.75 50.4318.13 370 0.00 2.84 0.42 78.17 18.57

[0225] When the % of remaining TZK was smaller than 3%, the reaction wasstopped. The reaction solution contained TZA with a HPLC purity of78.17%.

EXAMPLE 14 Synthesis of7-amino-3-[(1,2,5,6-tetrahydro-2-methyl-5,6-dioxo-1,2,4-triazin-3-yl)-thiomethyl]-cephalosporanicacid (TTA) in a single step

[0226] A filtrate (50 ml) from the strong anion exchanger Amberlite®IRA-400 containing 0.0016 moles of7β-(5-amino-5-carboxypentamido)-3-[(1,2,5,6-tetrahydro-2-methyl-5,6-dioxo-1,2,4-triazin-3-yl)-thiomethyl]cephalosporanicacid (hereinbelow indicated as TTC) with 91.1% purity (HPLC) and lessthan 0.2 mg/ml of2,5-dihydro-3-mercapto-2-methyl-5,6-dioxo-1,2,4-triazine (TTZ) wasadjusted to pH 7.25 with 3 M ammonia.

[0227] The TTC solution was fed into a 0.125 litre stirred glass vesselwith 16 g of wet Eupergit C250L with a co-immobilised D-amino acidoxidase/catalase system (25 U of DAAO/g and 30 kU of catalase/g) and 23g of wet Glutaryl-7-ACA Acylase (87 U/g).

[0228] The conversion was performed at 20° C., 400 rpm and with anoxygen flow through a bottom diffuser of 0.1 vol/vol/min at 1 barabsolute pressure. The pH was titrated to pH 7.25 with 3 M ammonia by anautotitrator.

[0229] The conversion was controlled by HPLC in a reverse phase columnEclipse XDB-C8 150 mm×4.6 mm ID×5μ; the mobile phase was 10 mMtetrabutylammonium hydrogen sulfate, 15 mM potassium dihydrogenphosphate, pH 6.5 containing 35% methanol at 1 ml/min with a 260 nmdetection. The TTC appeared at 2.4 minutes, the TTK at 6.1 min, the TTGat 5.5 min and the TTA at 2.9, respectively.

[0230] Representative samples of the reaction mixture except for theenzyme were taken and the result obtained are reported as percentage oftotal β-lactams in the following table: Time (min) % TTC % TTK % TTG %TTA % Side products 0 91.11 0.00 0.00 0.00 8.89 15 41.53 15.87 0.0036.27 6.33 60 0.00 6.79 0.00 80.38 12.83 120 0.00 0.00 0.00 88.69 11.31

[0231] When the % of remaining TTK was smaller than 3%, the reaction wasstopped. The reaction solution contained TTA with a HPLC purity of88.69%. The invention is not limited to the embodiments hereinbeforedescribed which may be varied in detail.

1. A process for preparing cephalosporanic acid derivatives comprisingthe steps of:— enzymatically converting a 3-thiolated cephalosporin Ccompound of formula III:—

 into a 3-thiolated-α-ketoadipyl-7-aminocephalosporanic acid derivativeof formula IV:

 wherein r is a heterocyclic group comprising at least a nitrogen atom.2. A process as claimed in claim 1 wherein the compound of formula IIIis enzymatically converted into a compound of formula IV by animmobilised enzyme system.
 3. A process as claimed in claim 2 whereinthe enzyme system comprises co-immobilised D-Amino acid oxidase andcatalase.
 4. A process as claimed in claim 3 wherein the enzymaticconversion is carried out in the presence of molecular oxygen, at apressure of 1 to 5 bar absolute, a pH of from 6.5 to 8.0 and at atemperature of from 15 to 30° C. for a period of from 30 mins to 180mins.
 5. A process as claimed in claim 1 comprising the step ofseparating the enzyme system from the reaction mixture, preferably byfiltration.
 6. A process as claimed in claim 1 including the step ofpurifying the compound of formula IV.
 7. A process as claimed in claim 6wherein the compound is purified using an adsorption column.
 8. Aprocess as claimed in claim 1 wherein the enzymes are co-immobilisedusing a suitable cross-linker agent in a suitable solid support.
 9. Aprocess as claimed in claim 8 wherein the enzymes are in the form ofcrystals of a size suitable for use as a biocatalyst.
 10. A process asclaimed in claim 1 wherein the enzymatic processes are carried out whilemaintaining the enzyme in dispersion in an aqueous substrate solution.11. A process as claimed in claim 1 wherein the or each enzymaticprocess is carried out in a column.
 12. A process as claimed in claim 1including the step of recovering the enzyme for reuse.
 13. A process asclaimed in claim 1 wherein the compound of formula IV is used withoutpurification in a continuous process for obtaining any usefulderivative.
 14. A process as claimed in claim 1 wherein R is aheterocyclic group comprising at least one nitrogen atom and optionallya sulphur or oxygen atom.
 15. A process as claimed in claim 14 wherein Ris a heterocyclic group selected from any one or more of the groupcomprising thienyl, diazolyl, tetrazolyl, thiazolyl, triazinyl,oxazolyl, oxadiazolyl, pyridyl, pirimidinyl, benzo thiazolyl,benzimidazolyl, benzoxazolyl, or any derivative thereof, preferably5-methyl-1,3,4-thiadiazol-2-yl, 1-methyl-1H-tetrazol-5-yl or1,2,5,6-tetrahydro-2-methyl-5,6-dioxo-1,2,4-triazin-3-yl.
 16. A3-thiolated-α-ketoadipyl-7-aminocephalosporanic acid derivative offormula IV whenever prepared by a process as claimed in claim
 1. 17. Acompound of the Formula:—

 wherein in formula IV, R is1,2,5,6-tetrahydro-2-methyl-5,6-dioxo-1,2,4-triazin-3-yl.
 18. A compoundof the Formula:—

 wherein in formula IV, R is 1-methyl-1H-tetrazol-5-yl.
 19. Use of acompound of formula IV as defined in claim 1 as an intermediate in aprocess for preparing cephalosporin C antibiotics.
 20. Use of anintermediate compound of the formula:—

in a process for preparing cephalosporin C antibiotics wherein informula IV R is 5-methyl-1,3,4-thiadiazol-2-yl.
 21. A process forpreparing cephalosporanic acid derivatives as claimed in claim 1comprising the step of: enzymatically converting a compound of formulaIV to form a compound of formula I

 wherein R is a heterocyclic group comprising at least one nitrogen atomand R₁ and R₂ are both hydrogen atoms or one of them is a hydrogen atomand the other is an acyl donor.
 22. A process as claimed in claim 21wherein a compound of formula IV is enzymatically converted to form acompound of formula I using Glutaryl-7-ACA acylase.
 23. A process asclaimed in claim 21 wherein the enzymation takes place at a temperatureof approximately 20° C. and at a pH of between 6.5 and 8.0.
 24. Aprocess as claimed in claim 21 wherein the enzyme is imrnmobilised usinga suitable cross-linker agent in a suitable solid support.
 25. A processas claimed in claim 24 wherein the enzyme is in the form of crystals ofa size suitable for use as a biocatalyst.
 26. A process as claimed inclaim 21 wherein enzymation is carried out while maintaining the enzymein dispersion in an aqueous substrate solution.
 27. A process as claimedin claim 21 wherein the enzymatic process is carried out in a column.28. A process as claimed in claim 21 including the step of recoveringthe enzyme for reuse.
 29. Use of a compound of formula I as defined inclaim 21 as an intermediate in a process for preparing cephalosporin Cderivatives.
 30. A process for preparing 3-thiolated cephalosporanicacid derivatives comprising the steps of;— enzymatically converting acompound of formula III

 into a 3-thiolated-α-ketoadipyl-7-aminocephalosporanic acid derivativeof formula IV:

 and enzymatically converting a compound of formula IV to form a3-thiolated 7-ACA compound of formula I

 wherein R is a heterocyclic group comprising at least one nitrogen atomand R₁ and R₂ are both hydrogen atoms or one of them is a hydrogen atomand the other is an acyl donor.
 31. A process as claimed in claim 30wherein the compound of formula III is enzymatically converted into acompound of formula I in one step by an immobilised enzyme system.
 32. Aprocess as claimed in claim 31 wherein the enzyme system comprises acombination of co-immobilised D-amino acid oxidase/catalase in thepresence of immobilised Glutaryl-7-ACA acylase.
 33. A process as claimedin claim 30 wherein the enzymation takes place at a temperature ofapproximately 20° C. and at a pH of between 6.5 and 8.0.
 34. A processas claimed in claim 30 wherein the enzymes are co-immobilised using asuitable cross-linker agent in a suitable solid support.
 35. A processas claimed in claim 34 wherein the enzymes are in the form of crystalsof a size suitable for use as a biocatalyst.
 36. A process as claimed inclaim 30 wherein the enzymatic processes are carried out whilemaintaining the enzyme in dispersion in an aqueous substrate solution.37. A process as claimed in claim 30 wherein the or each enzymaticprocess is carried out in a column.
 38. A process as claimed in claim 30including the step of recovering the enzyme for reuse.
 39. A process asclaimed in claim 30 wherein the compound of formula III is used withoutpurification in a continuous process for obtaining any usefulderivative.
 40. A process for preparing cephalosporanic acid derivativescomprising the steps of:— reacting cephalosporin C with a thiol compoundof the general formula II R—SH  II  wherein R is a heterocyclic groupcomprising at least one nitrogen atom, to form a 3-thiolatedcephalosporin Compound of formula III

 wherein R is as defined above, and, after formation of the compound offormula III removing excess thiol of formula II.
 41. A process asclaimed in claim 40 wherein the excess thiol is removed by adsorption onan anion exchange resin.
 42. A process as claimed in claim 41 whereinthe anion exchange resin is a microporous resin having a cross-linkedacrylic copolymer structure.
 43. A process as claimed in claim 42wherein the anion exchange resin comprises an 8% cross-linkingcontaining functional thialkyl benzyl ammonium group.
 44. A process asclaimed in claim 41 wherein the resin is in the chloride, hydroxy,phosphate or acetate cycle.
 45. A process as claimed in claim 40 whereinthe excess thiol is removed by crystallisation.
 46. A process as claimedin claim 45 wherein crystallisation is carried out at an acidic pH. 47.A process as claimed in claim 40 wherein the excess thiol is removed bycrystallisation followed by adsorption on an anion exchange resin.
 48. Aprocess as claimed in claim 40 wherein the cephalosporin C is in anaqueous medium.
 49. A process as claimed in claim 40 wherein thecephalosporin C is in the form of a concentrated cephalosporin Csolution.
 50. A process as claimed in claim 40 wherein the reaction iscarried out at a pH of between 5.5 and 8.0, at a temperature of from 60°C. to 80° C., for a period of from 1 to 8 hours.
 51. A process asclaimed in claim 40 wherein the reaction is carried out at a pH ofapproximately 6.0 and at a temperature of approximately 65° C.
 52. Aprocess as claimed in claim 40 wherein the thiol compound is present inan amount of between 1 and 5 mol/mol of cephalosporin C.
 53. A processas claimed in claim 40 wherein R is a heterocyclic group comprising atleast one nitrogen atom and optionally a sulphur or oxygen atom.
 54. Aprocess as claimed in claim 40 wherein R is a heterocyclic groupselected from any one or more of thienyl, diazolyl, thiazolyl,tetrazolyl, thiadiazolyl, triazinyl, oxazolyl, oxadiazolyl, pyridyl,pirimidinyl, benzothiazolyl, benzimidazolyl, benzoxazolyl, or anyderivative thereof, preferably 5-methyl-1,3,4-thiadiazol-2-yl,1-methyl-tetrazol-5-yl or1,2,5,6-tetrahydro-2-methyl-5,6-dioxo-1,2,4-triazin-3-yl.
 55. A compoundof formula III

wherein R is a heterocyclic group comprising at least one nitrogen atom,obtained by a process as claimed in any of claims 40 to
 54. 56. Acompound of the Formula:—

 wherein in formula III R is 5-methyl-1,3,4-thiadiazol-2-yl.
 57. Acompound of the Formula:—

 wherein in formula III R is1,2,5,6-tetrahydro-2-methyl-5,6-dioxo-1,2,4-triazin-3-yl.
 58. Use of acompound of formula III as defined in claim 55 as an intermediate in aprocess for preparing cephalosporin C derivatives.
 59. A process forpreparing cephalosporanic acid derivatives comprising the steps of:—enzymatically converting a 3-thiolated cephalosporin C compound offormula III obtained by a process as claimed in any of claims 40 to 54:—

 into a 3-thiolated-α-ketoadipyl-7-aminocephalosporanic acid derivativeof formula IV:

 wherein R is a heterocyclic group comprising at least a nitrogen atom.60. A process as claimed in claim 59 comprising the step of:enzymatically converting a 3-thiolated α-ketoadipyl 7-ACA compound offormula IV

 to form a 3-thiolated 7-ACA compound of formula I

 wherein R is a heterocyclic group comprising at least one nitrogen atomand R₁ and R₂ are both hydrogen atoms or one of them is a hydrogen atomand the other is an acyl donor.
 61. A process for preparingcephalosporanic acid dervatives comprising the step of:  enzymaticallyconverting a compound of formula IV

 to form a compound of formula I

 wherein R is a heterocyclic group comprising at least one nitrogen atomand R₁ and R₂ are both hydrogen atoms or one of them is a hydrogen atomand the other is an acyl donor.
 62. A process as claimed in claim 61wherein a compound of formula IV is enzymatically converted to form acompound of formula I with Glutaryl-7-ACA acylase.